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Ubiquitin vinyl sulfone, (HA-tag)

BML-UW0155-0025 25 µg 260.00 USD
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For use in the detection and identification of deubiquitinylating enzymes. HA-Ub-VS is a DUB active site directed probe generated by a chemical ligation method, that acts as a potent and irreversible inhibitor of DUBs through covalent modification of the active site and as a specific probe for enzymes with DUB activity. The HA peptide sequence (YPYDVPDYA), derived from the influenza hemagglutinin protein, facilitates sensitive identification or purification of HA-Ub-VS modified DUBs through recognition by anti-HA antibodies and/or anti-HA-agarose.

Product Specification

Alternative Name:HA-Ub-VS
UniProt ID:P0CG47 (UBB), P0CG48 (UBC), P62979 (RPS27A), P62987 (UBA52)
Purity:≥95% (SDS-PAGE)
Application Notes:Suggested uses:
1. Deubiquitinylating enzyme active site-directed probes for detection of novel DUBs.
2. Determination of deubiquitinylating enzyme activity of putative DUBs.
3. Identification of DUBs from biological samples via immunoprecipitation and proteomic methods.
4. Characterization of deconjugating enzymes for Ub/Ubl specificity.
Reconstitution:Reconstitute with aqueous buffer up to 5mg/ml (~500µM).
Shipping:Shipped on Dry Ice
Short Term Storage:-80°C
Long Term Storage:-80°C
Use/Stability:Stable for at least 6 months after receipt when stored at -80°C. Once reconstituted store HA-Ub-VS solution at -80°C, avoid multiple freeze/thaw cycles and use within 1 month.
Handling:Avoid freeze/thaw cycles.
Technical Info/Product Notes:Typical assay set-up:HA-Ub-VS probe concentration: 0.5-5.0μM. Purified enzyme concentration: 0.1-1.0µM; Buffer: 50mM TRIS, pH7.5, 150mM NaCl, 1mM DTT. Incubate at 37°C for 30 minutes. Modification of DUB enzymes by site-directed HA-Ub-VS probes detected by Western blotting with either HA antibody (general reactivity) or an antibody specific to particular DUB of interest. Note: When probing complex solutions such as cell lysates the optimal amount of total sample protein required will depend on the specific lysate to be analyzed and must therefore be determined for the individual lysate of interest.
bml-uw0155 dub rxns-5
Image: DUB active site probe assay: Western blot showing reactions containing HAUbVS only (lane 1), HAUbVS + GST-UCHL1 (lane 2, Prod. No. BML-UW9305), and HA-UbVS + USP2cd (lane 3, Prod. No. BML-UW9850). HAUbVS modified proteins detected using HA polyclonal antibody (Sigma – H6908) at 1:1000 dilution.
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bml-uw0155 dub rxns-5

Product Literature References

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples: P. Griffin, et al.; J. Vis. Exp. e52784 (2015), Abstract;

General Literature References

Derivitization of the C-terminus of ubiquitin and ubiquitin-like proteins using intein chemistry: methods and uses: K.D. Wilkinson, et al.; Methods Enzymol. 399, 37 (2005), Abstract;
Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family: A. Borodovsky, et al.; Chem. Biol. 9, 1149 (2002), Abstract;
A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14: A. Borodovsky, et al.; EMBO J. 20, 5187 (2001), Abstract;

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